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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3


TITLE: QUALITY CONTROLS


Contamination of cell cultures can lead to an array of problems, many of which can be underestimated. The damage caused to cell lines in culture by bacteria and fungi contamination is widely recognised and normally easy to see. In contract the effects of mycoplasma can be wide ranging and less obvious including: a reduction in growth rate, induction of morphological changes, chromosomal aberrations altering cell metabolism and loss of cell recovery following cryopreservation.

 All the cell lines proposed by CABRI are tested to verify freedom from contamination by mycoplasma, bacteria, yeast, fungi and virus. In addition, the cell lines undergone tests to confirm species of origin and tests for individual identification.

 

FLOW DIAGRAM

GENERAL CONSIDERATIONS ON MYCOPLASMA

 

Minimum QC requirements

(A) Preliminary Screen

Viability Check

  • Cell count and viability:
    - vital dye exclusion (trypan blue)
    AHC/1998/3/3.1/2.1
    Acceptance criteria: > 2 x 106 cells/vial, > 80% viability, vigorous cell growth

Microbial QC

  • Mycoplasma testing

- Hoechst stain/DAPI stain, AHC/1998/3/3.2/2.1
- Isolation by culture

Acceptance criteria - all tests negative

(B) Full QC and Authentication Screen Master cell banks

Viability Check

Acceptance criteria - > 2 x 106 cells/vial, > 80% viability, vigorous cell growth

Microbial QC

  • Mycoplasma testing

- Hoechst stain/DAPI stain, AHC/1998/3/3.2/2.1
- Isolation by culture,
AHC/1998/3/3.2/2.2

  • Bacteria/fungi testing

- Isolation by culture, AHC/1998/3/3.3/2.1

Acceptance criteria - all tests negative

Authentication Testing

  • Isoenzyme analysis

Acceptance criteria - species confirmed as correct

Additional testing is carried out at individual centres as indicated below.

Authentication Assays

Microbial QC

Virus Screening

 

(C) Full QC and Authentication Screen Distribution cell banks

Viability Check

  • Cell count and viability:

- vital dye exclusion (trypan blue), AHC/1998/3/3.1/2.1
Acceptance criteria - > 2 x 106 cells/vial, > 80% viability, vigorous cell growth

Microbial QC

  • Mycoplasma testing

- Hoechst stain/DAPI stain: AHC/1998/3/3.2/2.1

- Isolation by culture

  • Bacteria/fungi testing

- Isolation by culture: AHC/1998/3/3.3/2.1

Acceptance criteria - all tests negative

Additional testing is carried out at individual centres as indicated below.

Authentication Assays

 

Microbial QC

 


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

© The CABRI Consortium 1999-2013.
This work cannot be reproduced in whole or in part without the express written permission of the CABRI consortium.
Site maintained by Paolo Romano. Last revised on April 2013.